1. Field of the Invention
The present invention relates to superoxide dismutase derivatives.
The superoxide dismutase derivatives provided by the present invention retain most of the enzymatic activities of superoxide dismutase capable of decomposing superoxide, which is harmful to living organisms, and have an extremely prolonged plasma half-life as compared with the superoxide dismutase. Also, the superoxide dismutase derivatives of the present invention are effective for treating various diseases caused by active oxygen species, and can be used as anti-inflammatory agents, anti-ulcer agents, anti-ischemic agents, cerebral edema-treating agents and paraquat intoxication-treating agents. Further, the superoxide dismutase derivatives are useful for drugs for alleviating various side effects induced by anti-cancer agents, as caused by active oxygen species.
The present invention also relates to chemical modifiers to provide the above-mentioned superoxide dismutase derivatives.
2. Description of the Prior Art
Superoxide dismutase (hereinafter sometimes referred to as "SOD") is known as an enzyme occurring widely in living organisms, such as animals, plants and microorganisms, and decomposing superoxide which is harmful to living organisms.
In recent years, attempts have been made to use SOD as an anti-inflammatory agent [Farumashia, 17, 411-412 (1981); Current Therapeutic Research, 16, 706 (1974)]; as a protective agent for cardiac surgery [Furii Rajikaru no Rinsho (Clinical Medicine on Free Radical), Vol. 5, 113-119, Nihon Igakukan, issued on Nov. 30, 1990]. It is said that the plasma half-life of intravenously administered SOD is only 4 to 6 minutes; that is, SOD is rapidly metabolized and excreted into urine since the molecular weight of SOD (about 32,000) is smaller than the glomerular filtration limit (corresponding to a molecular weight of about 50,000). For the purpose of prolonging the plasma half-life of SOD, attempts have been made to modify SOD with natural polymers and synthetic organic compounds.
SOD derivatives modified with natural polymers such as Ficoll, inulin, dextran sulfate, dextran, heparin, hyaluronic acid, and glycosaminoglycan are known [Proc. Natl. Acad. Sci. USA, 77, 1159 (1980); Japanese Patent Application Laid-open Nos. 32826/1983, 231075/1990, 231076/1990, 231077/1990, 231078/1990 and 273176/1990]. However, these SOD derivatives do not have a definite molecular weight since each of the natural polymers has a certain distribution in its molecular weight. Rat albumin-modified SOD has also been reported, but it has antigenicity [Agents and Actions, 10, 231 (1980)].
SOD derivatives modified with synthetic polymers such as polyethylene glycol and polyalkylene glycol are known as well [Res. Commun. Chem. Pathol. Pharmacol., 29, 113 (1980); Japanese Patent Application Laid-open No. 249388/1986]. However, each of the synthetic polymers also has a certain molecular weight distribution.
Modification of SOD with a partially half-esterified styrene-maleic anhydride copolymer (hereinafter referred to as "SMA") has been investigated [Japanese Patent Application Laid-open No. 104164/1989]. The SOD derivative modified with SMA (hereinafter referred to as "SMA-SOD") has a prolonged plasma half-life because it binds reversibly plasma protein, especially albumin, and further accumulates, due to the presence of carboxylic acid groups in SMA, in regions made acidic by inflammation. Other SOD derivatives in which two SOD molecules are crosslinked via a dicarboxylic acid and which have a narrower molecular weight distribution are also known [Japanese Patent Application Laid-open No. 95775/1989], but they can hardly be expected to accumulate in inflammatory regions.
All of the above SOD derivatives have been modified by using the amino groups of SOD. Human-type SOD has 22 or 24 amino groups per molecule, while bovine-type SOD has 20 amino groups per molecule. Since SOD thus has a number of amino groups, it is very difficult to specify which amino groups of SOD have reacted with the modifier used. In view of the current situation in which the compound to be used as a medicinally active ingredient should preferably have a definite single chemical structure, it is preferred that SOD derivatives to be used for medicinal purposes should have been modified at specific sites in the molecule thereof.
From these points of view, an SOD derivative modified, via its mercapto groups, by an SMA modified with a functional group reactive with mercapto group (hereinafter referred to as "SMI-SOD") has been prepared [Japanese Patent Application Laid-open No. 257479/1989]. The plasma half-life, enzymatic activities and accumulation property in acidic regions of this SOD derivative are similar to those of above-described SMA-SOD. Moreover, this SOD derivative has definite modification sites in the SOD molecule. However, the structure of SMA is, although specified from the viewpoint of polymer chemistry, still complicated and the SMI-SOD therefore has problems in view of the above-described utility as a medicinally active ingredient.
Accordingly, there has been strongly demanded development of an SOD derivative whose plasma half-life, enzymatic activities and accumulation property in acidic regions are similar to those of SMA-SOD and SMI-SOD, and which is modified at specific sites in the SOD molecule and with a modifier having a definite chemical structure.
It is an object of the present invention to provide a novel SOD derivative which is prepared by bonding a long chain carboxylic acid maleimide to each of two mercapto groups in the SOD molecule and has a significantly prolonged plasma half-life as compared with that of unmodified SOD, the characteristic of accumulating in acidic regions and a definite single chemical structure.
It is another object of the invention to provide a novel long chain carboxylic acid maleimide useful as a chemical modifier for SOD.